EP0907660A1 - Gene scarecrow, son promoteur et ses utilisations - Google Patents

Gene scarecrow, son promoteur et ses utilisations

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Publication number
EP0907660A1
EP0907660A1 EP97928623A EP97928623A EP0907660A1 EP 0907660 A1 EP0907660 A1 EP 0907660A1 EP 97928623 A EP97928623 A EP 97928623A EP 97928623 A EP97928623 A EP 97928623A EP 0907660 A1 EP0907660 A1 EP 0907660A1
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European Patent Office
Prior art keywords
leu
ser
ala
scr
glu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP97928623A
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German (de)
English (en)
Other versions
EP0907660A4 (fr
Inventor
Philip N. Benfey
Laura Dilaurenzio
Joanna Wysocka-Diller
Jocelyn E. Malamy
Leonard Pysh
Yrjo Helariutta
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New York University NYU
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New York University NYU
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Publication of EP0907660A1 publication Critical patent/EP0907660A1/fr
Publication of EP0907660A4 publication Critical patent/EP0907660A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8227—Root-specific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention generally relates to the SCARECROW (SCR) gene family and their promoters.
  • the invention more particularly relates to ectopic expression of members of the SCARECROW gene family in transgenic plants to artificially modify plant structures.
  • the invention also relates to utilization of SCARECROW promoter for tissue and organ specific expression of heterologous gene products.
  • Asymmetric cell divisions in which a cell divides to give two daughters with different fates, play an important role in the development of all multicellular organisms.
  • the regulation of asymmetric cell divisions is of heightened importance in determining organ morphology.
  • organs are not formed during embryogenesis. Rather, cells that form the apical meristems are set aside at the shoot and root poles. These reservoirs of stem cells are considered to be the source of all post- embryonic organ development in plants.
  • a fundamental question in developmental biology is how meristems function to generate plant organs. 2.1. ROOT DEVELOPMENT
  • Root organization is established during embryogenesis. This organization is propagated during postembryonic development by the root meristem. Following 5 germination, the development of the postembryonic root is a continuous process, a series of initials or stem cells continuously divide to perpetuate the pattern established in the embryonic root (Steeves & Hampshire, 1972, Patterns in Plant Development. Englewood Cliffs, NJ: Prentice-Hall, Inc.). 0 Due to the organization of the Arabidopsis root it is possible to follow the fate of cells from the meristem to maturity and identify the progenitors of each cell type (Dolan et al., 1993, Development 119:71-84). The Arabidopsis root is a relatively simple and well characterized organ. 5 The radial organization of the mature tissues in the
  • Arabidopsis root has been likened to tree rings with the epidermis, cortex, endodermis and pericycle forming radially symmetric cell layers that surround the vascular cylinder (FIG. IA) . See also Dolan et al., 1993, Development 0 119:71-84. These mature tissues are derived from four sets of stem cells or initials: i) the columella root cap initial; ii) the pericycle/vascular initial; iii) the epidermal/lateral root cap initial; and iv) the cortex/endodermal initial (Dolan et al., 1993, Development 25 119:71-84).
  • the cortex/endodermal initial for example, first divides anticlinally (in a transverse orientation) (FIG. IB) .
  • This asymmetric division produces another initial 30 and a daughter cell.
  • the daughter cell expands and then divides periclinally (in the longitudinal orientation) (FIG. IB) .
  • This second asymmetric division produces the progenitors of the endodermis and the cortex cell lineages (FIG. IB) . 35 2.2.
  • the cortex/endodermal initial divides anticlinally, but the subsequent periclinal division that increases the number of cell layers does not take place (Benfey et al., 1993, Development 119:57-70; Scheres et al., 1995, Development 121:53-62) .
  • the defect is first apparent in the embryo and it extends throughout the entire embryonic axis which includes the embryonic root and hypocotyl (Scheres et al., 1995, Development 121:53-62).
  • Gravitropic mutants have been studied for evidence that proves the role of amyloplast sedimentation in gravity sensing.
  • many gravitropic mutations affect downstream events such as auxin sensitivity or metabolism (Masson, 1995, BioEssays 17:119-127).
  • Other mutations seem to affect gene products that process information from gravity sensing.
  • the lazy mutants of higher plants and comparable mutants in mosses can clearly sense and respond to gravity, but the mutations reverse the normal polarity of the gravitropic response (Gaiser & Lomax, 1993, Plant Physiol. 102:339-344; Jenkins et al., 1986, Plant Cell Environ 9:637- 644) .
  • Other mutations appear to affect gravitropism of specific organs.
  • sgr mutants have defective shoot gravitropism (Fukaki et al., 1996, Plant Physiol. 110:933-943; Fukaki et al., 1996, Plant Physiol. 110:945-955; Fukaki et al., 1996, Plant Res. 109:129-137).
  • SCR SCARECROW
  • the invention relates to the SCARECROW (SCR) gene (which encompasses the Arabidopsis SCR gene and its orthologs and paralogs) , SCR gene products, (including but not limited to transcriptional products such as mRNAs, antisense and ribozyme molecules, and translational products such as the SCR protein, polypeptides, peptides and fusion proteins related thereto) , antibodies to SCR gene products, SCR regulatory regions and the use of the foregoing to improve agronomically valuable plants.
  • SCR SCARECROW
  • the invention is based, in part, on the discovery, identification and cloning of the gene responsible for the scarecrow phenotype.
  • the inventors have determined that the mutant cell layer in roots of ser mutants has differentiated characteristics of both cortex and endodermis. This is consistent with a role for SCR in the regulation of the asymmetric cell division rather than in specification of the identity of either cortex or endodermis.
  • the inventors have also determined that SCR expression affects the gravitropism of plant aerial structures such as the stem.
  • One aspect of the invention relates to the heterologous expression of SCR genes and related nucleotide sequences, and specifically the Arabidopsis SCR genes, in stably transformed higher plant species. Modulation of SCR expression levels can be used to advantageously modify root and aerial structures of transgenic plants and enhance the agronomic properties of such plants.
  • Another aspect of the invention relates to the use of promoters of SCR genes, and specifically the use of Arabidopsis SCR promoter to control the expression of protein and RNA products in plants.
  • Plant SCR promoters have a variety of uses, including but not limited to expressing heterologous genes in the embryo, root, root nodule, and stem of transformed plants.
  • the invention is illustrated by working examples described infra which demonstrate the isolation of the Arabidopsis SCR gene using insertion mutagenesis. More specifically, T-DNA tagging of genomic and cDNA clones of the Arabidopsis SCR gene are described. Additional working examples include the isolation of SCR sequences from plant genomes using PCR amplification in combination with screening of genomic libraries, and heterologous gene expression in transgenic plants using SCR promoter expression constructs. Structural analysis of the deduced amino acid sequence of Arabidopsis SCR protein indicates that SCR encodes a transcription factor. Northern analysis, in situ hybridization analysis and enhancer trap analysis show highly localized expression of Arabidopsis SCR in embryos and roots. Genetic analysis shows SCJ? expression also affects gravitropism of aerial structures (e . g. , stems). This indicates that SCR is also expressed in those structures.
  • CDNA complementary DNA cis-regulatory element A promoter sequence 5' upstream of the TATA box that confers specific regulatory response to a promoter containing such an element.
  • a promoter may contain one or more cis- regulatory elements, each responsible for a particular regulatory response coding sequence sequence that encodes a complete or partial gene product (e .g. , a complete protein or a fragment thereof)
  • EST expression tagged functional portion a functional portion of a promoter is any portion of a promoter that is capable of causing transcription of a linked gene sequence, e .g. , a truncated promoter
  • gene fusion a gene construct comprising a promoter operably linked to a heterologous gene, wherein said promoter controls the transcription of the heterologous gene gene product the RNA or protein encoded by a gene sequence gene sequence sequence that encodes a complete gene product (e . g . , a complete protein)
  • GUS 1,3- ⁇ -Glucuronidase gDNA genomic DNA heterologous gene
  • a heterologous gene means that the gene is linked to a promoter that said gene is not naturally linked to.
  • the heterologous gene may or may not be from the organism contributing said promoter.
  • maize ZCARECROW gene is an ortholog of the Arabidopsis SCR gene) paralog related gene in the same plant (e . g . , Arabidopsis SRPal is a paralog of Arabidopsis SCR gene)
  • SCR SCARECROW gene or gene product encompasses (italic) SCR and ZCR genes and their orthologs and paralogs
  • ZCR maize ZCARECROW gene, a paralog of, for example, the Arabidopsis SCR gene
  • SCR protein means a protein containing sequences or a domain substantially similar to one or more motifs (i.e., Motif I-VI) , preferably MOTIF III (VHIID) , of Arabidopsis SCR protein as shown in FIGS. 13A-F and FIGS. 15A-S.
  • SCR proteins include SCR ortholog and paralog proteins having the structure and activities described herein.
  • SCR polypeptides and peptides include deleted or truncated forms of the SCR protein, and fragments corresponding to the SCR motifs described herein.
  • SCR fusion proteins encompass proteins in which the SCR protein or an SCR polypeptide or peptide is fused to a heterologous protein, polypeptide or peptide.
  • SCR gene, nucleotides or coding sequences means nucleotides, e.g., gDNA or cDNA encoding SCR protein, SCR polypeptides or peptides, or SCR fusion proteins.
  • SCR gene products include transcriptional products such as mRNAs, antisense and ribozyme molecules, as well as translational products of the SCJ? nucleotides described herein including but not limited to the SCR protein, polypeptides, peptides and/or SCR fusion proteins.
  • SCJ? promoter means the regulatory region native to the SCR gene in a variety of species, which promotes the organ and tissue specific pattern of SCJ? expression described herein.
  • FIGS. 1A-B Schematic of Arabidopsis root anatomy.
  • FIG. IA Transverse section showing the four tissues, epidermis, cortex, endodermis and pericycle that surround the vascular tissue.
  • the epidermal/lateral root cap initials and the cortex/endodermal initials are shown at the base of their respective cell files.
  • FIG. IB Schematic of division pattern of the cortex/endodermal initial. The initial expands then divides anticlinally to reproduce itself and a daughter cell. The daughter then divides periclinally to produce the progenitors of the endodermis and cortex cell lineages.
  • C cortex
  • Da daughter cell
  • E endodermis
  • In initial.
  • FIGS. 2A-F Phenotype of scr mutant plants.
  • FIG. 2A Shown left to right are 12-day scr-2, scr-1 and wild-type seedlings grown vertically on nutrient agar medium.
  • FIG. 2B 21-day scr-2 mutant plants in soil.
  • FIG. 2C The third section.
  • FIG. 2D Transverse section through primary root of 7-day scr-2.
  • FIG. 2D Transverse section through primary root of 7-day wild- type (WT) .
  • FIG. 2E Transverse section through lateral root of 12-day scr-1 mutant seedling.
  • FIG. 2F Transverse section through root regenerated from scr-l callus. Bar, 50 ⁇ m. Abbreviations: C, cortex; En, endodermis; Ep, epidermis; M, mutant cell layer; P, pericycle; V, vascular tissue.
  • FIGS. 3A-F Characterization of the cellular identity of the mutant cell layer.
  • FIG. 3A Endodermis- specific Casparian band staining of transverse sections through the primary root of 7-day scr-1 mutant.
  • FIG. 3B Casparian band staining of transverse sections through the primary root of 7-day wild-type (WT) .
  • FIG. 3C Immunostaining with the endodermis (and a subset of vascular tissue) specific JIM13 monoclonal antibodies on transverse root sections of scr-2 mutant.
  • FIG. 3D The endodermis (and a subset of vascular tissue) specific JIM13 monoclonal antibodies on transverse root sections of scr-2 mutant.
  • FIG. 3E Immunostaining with the JIM7 monoclonal antibody that stains all cell walls on transverse root sections of scr-2 mutant.
  • FIG. 3F Immunostaining with JIM7 monoclonal antibodies on transverse root sections of WT. Bar, 25 ⁇ m. Abbreviations are same as those for description of FIGS. 2A-2F and: Ca, casparian strip.
  • FIGS. 4A-F Immunostaining.
  • FIG. 4A Immunostaining with the cortex (and epidermis) specific CCRC- M2 monoclonal antibodies on transverse root sections of scr-1 mutant.
  • FIG. 4B Immunostaining with CCRC-M2 antibodies on transverse root sections of scr-2 mutant.
  • FIG. 3C Immunostaining with CCRC-M2 antibodies on transverse root sections of wild-type (WT) .
  • FIG. 4D Immunostaining with the CCRC-M1 monoclonal antibodies (specific to a cell wall epitope found on all cells) on transverse root sections of scr-1.
  • FIG. 4E Immunostaining with CCRC-M1 antibodies on transverse root sections of scr-2 .
  • FIG. 4F Immunostaining with CCRC-M1 antibodies on transverse root sections of WT. Bar, 30 ⁇ m. Abbreviations are same as those for description Of FIGS. 2A-2F.
  • FIG. 5A-E Structure of the Arabidopsis SCARECROW gene.
  • FIG. 5A Nucleic acid sequence and deduced amino acid sequence of the Arabidopsis SCR genomic region (SEQ ID NO:l) and (SEQ ID N0:2), respectively. Regulatory sequences including: (i) TATA box, (ii) ATG start codon, and (iii) potential polyadenylation sequence are underlined. Within the deduced amino acid sequence homopolymeric repeats are • underlined.
  • FIG. 5B Schematic diagram of genomic clone indicating possible functional motifs, T-DNA insertion sites and subclones used as probes.
  • FIG. 5C Comparison of the charged region found in Arabidopsis SCR protein with that found in bZIP transcription factors, SCR bZIP-like domain (SEQ ID NO:3), GCN4 (SEQ ID NO:3), SEQ ID NO:3
  • FIG. 5D TGAl (SEQ ID N0:5), C-Fos (SEQ ID NO:6), C-JUN (SEQ ID NO:7), CREB (SEQ ID NO:8), Opaque-2 (SEQ ID NO:9), OBF2 (SEQ ID NO:10), RAF-1 (SEQ ID NO:11).
  • FIG. 5D TGAl (SEQ ID N0:5), C-Fos (SEQ ID NO:6), C-JUN (SEQ ID NO:7), CREB (SEQ ID NO:8), Opaque-2 (SEQ ID NO:9), OBF2 (SEQ ID NO:10), RAF-1 (SEQ ID NO:11).
  • FIGS. 6A-B Expression of the Arabidopsis SCARECROW gene.
  • FIG. 6A Northern blot of total RNA from wild-type siliques (Si) , roots (R) , leaves (L) and whole seedlings (Sd) hybridized with Arabidopsis SCR probe a and with a probe from the Arabidopsis glutamine dehydrogenase (GDH) gene (Melo-Oliveira et al., 1996, Proc. Natl. Acad. Sci. USA 93:4718-4723) as a control for RNA integrity.
  • GDH Arabidopsis glutamine dehydrogenase
  • FIG. 6B Northern blot of Arabidopsis wild-type, scr-1 and scr-2 total RNA, probed with Arabidopsis SCR probe "a" corresponding to a cDNA sequence shown in FIG. 5B, and with the GDH probe. In scr-2 mutant additional bands of 4.1 kb and 5.0 kb were detected.
  • FIGS. 7A-G In situ hybridization and enhancer trap analyses of Arabidopsis SCR expression.
  • FIG. 7A SCR RNA expression detected by in situ hybridization of SCJ? antisense probe to a longitudinal section through the root meristem.
  • FIG. 7B In_ situ hybridization of SCR antisense probe to a transverse section in the meristematic region.
  • FIG. 7C In situ hybridization of SCJ? antisense probe to late torpedo stage embryo.
  • FIG. 7D Negative control in situ hybridization using a SCR sense probe to a longitudinal section through the root meristem.
  • FIG. 7E GUS expression in a whole mount in the enhancer trap line, ET199 in primary root tip.
  • FIG. 7F GUS expression in a whole mount in the enhancer trap line, ET199 in primary root tip.
  • FIG. 7G GUS expression in ET199 detected in a section through the root meristem. GUS expression is observed in the cortex/endodermal initial, and in the first cell in the endodermal cell lineage but not in the first cell of the cortex lineage. Expression in two endodermal layers is observed higher up in the root because the section was not median at that point. Bar, 50 ⁇ m. Abbreviations are same as those in the description of FIGS. 2A-2F.
  • FIG. 9 Partial nucleotide sequence (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:21) of the Arabidopsis SRPa3 gene.
  • FIG. 10 Partial nucleotide sequence (SEQ ID NO:22) of the Arabidopsis SRPal gene.
  • FIG. IIB Partial nucleotide sequence (SEQ ID NO:25) and deduced amino acid sequence (SEQ ID NO:26) of the maize SRPml gene (Zm-Scl2) .
  • FIG. 12A-B Nucleotide sequence of rice SRPo3 EST clone.
  • FIG. 12A Sequence of 5' end of EST clone (SEQ ID N0:28) .
  • FIG. 12B Sequence of 3' end of EST clone (SEQ ID N0:29) .
  • FIGS. 13A-F Comparison of the amino acid sequence of members of the SCARECROW family of genes. conserveed Motifs I through VI are indicated by dashed line above the aligned sequences. Consensus sequences are shown in bold. See Table 1 for the identity and sequence identifier number of each of the sequences shown in this Figure.
  • Hu-scr-1 Human SCR paralog (SEQ ID NO:40).
  • FIG. 14 Restriction map of the approximately 8.8 kb Eco RI insert DNA of lambda clone, t643, containing the Arabidopsis SCR gene. The locations of the approximately 5.6 kb Hindlll-Sacl fragment subcloned in plasmid LIG l-3/SAC+MoB 2 1SAC, and the SCJ? coding region are indicated below the restriction map. The location of the translational initiation site of the SCR gene is at the Nco I site at the left end of the indicated coding region. The SCR coding sequence begins at the translation initiation site and extends approximately 1955 nucleotides to its right. E.
  • FIGS. 15A-S Comparison of the partial and complete amino acid sequences of several plant members of the SCARECROW family of genes. The amino acid sequences are aligned in a manner that maximizes amino acid sequence similarity and identity among SCR family members. Each sequence shown is continuous except where noted otherwise; the dots are inserted between two sequence segments in order to align homologous segments. "X" in the middle of a sequence indicates ambiguity in the corresponding nucleotide sequence and, possible termination of the ORF at the M X" residue site.
  • X at the end of a sequence indicates termination of the ORF at the "X" residue site.
  • the numbering of the amino acid residues is shown at the bottom of each figure and is based on the Arabidopsis SCR amino acid sequence. conserveed Motifs I through VI are indicated by the various dashed lines above the figures.
  • the new and old names of the family members are shown in FIG. 15A.
  • the sequences of SCR, Tfl and Tf4 are of the complete SCR protein. See Table l for the identity and the sequence identifier number of each sequence shown in these figures.
  • FIGS. 16A-M The partial nucleotide sequences of several plant members of the SCARECROW family of genes. M N" indicates an unknown base. See Table 1 for the identity and the sequence identifier number of each sequence shown in these figures.
  • FIG. 17A The partial nucleotide sequence (SEQ ID NO:66) of the maize ZCR gene.
  • FIG. 17B The partial amino acid sequence (SEQ ID NO:66) of the maize ZCR gene.
  • FIG. 19 Comparison of promoter activities in transgenic lines and roots.
  • Panel a A stably transformed line containing four copies of the B2 subdomain of the 35S promoter of CaMV upstream of GUS (Benfey et al., 1990). GUS is expressed in the root tip.
  • Panel b Roots emerging from callus transformed with four copies of the B2 subdomain of the 35S promoter fused to GUS. GUS expression can be seen in the emerging root tips (arrows) .
  • Panel c Higher magnification of a root emerging from the callus in panel b. GUS is clearly restricted to the root tip. The morphology of roots regenerated from calli often appears abnormal.
  • Panel d A transgenic plant regenerated from the calli and roots shown in panel b.
  • GUS expression in this plants appears to be similar to that of the original line shown in panel a.
  • Panel e. ET199 a stably transformed line that contains an enhancer trapping construct with a minimal promoter fused to the GUS coding region inserted 1 kb upstream from the SCR coding region.
  • GUS expression is primarily in the endodermal layer of the root.
  • GUS expression is primarily found in the endodermal layer as in ET199. The expression of GUS in the quiescent center, as seen here, is also sometimes observed in ET199. Bar, 50 ⁇ m.
  • FIG. 20 Analysis of SCJ? promoter activity in the scr mutant background.
  • Panel a Roots emerging from scr calli transformed with the SCR promoter::GUS construct. Roots regenerated from scr calli are very short. GUS expression appears to be limited to an internal layer of the root (arrows) .
  • Panel b Root regenerated from transformed scr calli and transferred to liquid culture. The scr phenotype, a single layer between the epidermis and pericycle, is easily seen. GUS expression is limited to this mutant layer.
  • E Epidermis.
  • M Mutant Layer.
  • P Pericycle. Bar, 50 ⁇ m.
  • FIG. 21 Molecular Complementation of the scr mutant.
  • FIG. 22 Expression of ZCR in maize root tips.
  • Left Panel Expression of ZCR is in the endodermal layer and extends down through the region of the quiescent center.
  • Right Panel Higher magnification showing expression in a single cell layer through the quiescent center.
  • the invention relates to the SCAJ?J?CJ?CW (SCR) gene, SCR gene products, including but not limited to transcriptional products such as mRNAs, antisense and ribozyme molecules, and translational products such as the SCR protein, polypeptides, peptides and fusion proteins related thereto; antibodies to SCR gene products; SCR regulatory regions; and the use of the foregoing to improve agronomically valuable plants.
  • SCR SCAJ?J?CJ?CW
  • the SCR genes and promoters of the present invention have a number of important agricultural uses.
  • the SCR promoters of the invention may be used in expression constructs to express desired heterologous gene products in the embryo, root, root nodule, and starch sheath layer in stem of transgenic plants transformed with such constructs.
  • SCR promoters may be used to express disease resistance genes such as lysozymes, cecropins, maganins, or thionins for anti-bacterial protection or the pathogenesis- related (PR) proteins such as glucanases and chitinases for anti-fungal protection.
  • SCR promoters also may be used to express a variety of pest resistance genes in the aforementioned plant structures and tissues.
  • Examples of useful gene products for controlling nematodes or insects include Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, chitinase, glucanases, lectins, and glycosidases.
  • SCJ? Gene constructs that express or ectopically express SCJ?, and the SCJ?-suppression constructs of the invention may be used to alter the root and/or stem structure, and the gravitropism of aerial structures of transgenic plants. Since SCR regulates root cell divisions, overexpression of SCJ? can be used to increase division of certain cells in roots and thereby form thicker and stronger roots. Thicker and stronger roots are beneficial in preventing plant lodging. Conversely, suppression of SCR expression can be used to decrease cell division in roots and thereby form thinner roots. Thinner roots are more efficient in uptake of soil nutrients. Since SCR affects gravitropism of aerial structures, overexpression of SCR may be used to develop "straighter" transgenic plants that are less susceptible to lodging.
  • SCR gene sequence may be used as a molecular marker for a qualitative trait, e . g . , a root or gravitropism trait, in molecular breeding of crop plants.
  • a qualitative trait e . g . , a root or gravitropism trait
  • the invention is described in the subsections below in terms of (a) SCR genes and nucleotides; (b) SCJ? gene products; (c) antibodies to SCJ? gene products; (d) SCJ? promoters and promoter elements; (e) transgenic plants which ectopically express SCR; (f) transgenic plants in which endogenous SCR expression is suppressed; and (g) transgenic plants in which expression of a transgene of interest is controlled by SCR promoter.
  • the SCARECROW genes and nucleotide sequences of the invention include: (a) a gene listed below in Table 1 (hereinafter, a gene comprising any one of the nucleotide sequences shown in FIG. 5A, FIG. 8, FIG. 9, FIG. 10, FIGS.
  • any gene comprising nucleotide sequence that hybridizes to the complement of any one of the sequences listed by their sequence identifier numbers in Table 1 or any segment of such nucleotide sequences, or as contained in any one of the clones described herein and deposited with the ATCC, under the following low stringency conditions: pre- hybridization in hybridization solution (HS) containing 43% formamide, 5xSSC, 1% SDS, 10% dextran sulfate, 0.1% sarkosyl, 2% block (Genius kit, Boehringer-Mannheim) , followed by hybridization overnight at 30 to 33°C using as a probe a DNA molecule of approximately 1.6 kb of SEQ ID NO:l at a concentration of 20 ng/ml, followed by washing in 2xSSC/0.1% S
  • any gene comprising nucleotide sequence that encodes a polypeptide or protein containing the consensus sequence for SCR (i.e., MOTIF III or VHIID) shown in FIGS. 13B-D or a segment of such polypeptide or protein.
  • SCR SCR
  • the partial and complete nucleotide and amino acid sequences of SCR genes and encoded proteins and polypeptides included in the invention are listed in Table 1 below.
  • Each EST clone is identified by its GenBank accession number. Each EST clone corresponds to a deposit of a cDNA sequence that matches a part of the nucleotide sequence of the corresponding SCJ? ortholog or paralog.
  • Functional equivalents of the SCR gene product include any plant gene product that regulates plant embryo or root development, or, preferably, that regulates root cell division or root tissue organization, or affects gravitropism of plant aerial structures (e.g., stems and hypocotyls) .
  • Functional equivalents of the SCR gene product include naturally occurring SCR gene products, and mutant SCJ? gene products, whether naturally occurring or engineered.
  • the invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of the nucleotide sequences (a) through (f) , in the first paragraph of this section.
  • Such hybridization conditions may be highly stringent, less highly stringent, or low stringency as described above.
  • highly stringent conditions may refer, e.g., to washing in 6xSSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos) , and 60°C (for 23-base oligos) .
  • nucleic acid molecules may act as SCR antisense molecules, useful, for example, in SCR gene regulation and/or as antisense primers in amplification reactions of SCR gene and/or nucleic acid sequences. Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also useful for SCR gene regulation. Still further, such molecules may be used as components in probing methods whereby the presence of a SCARECROW allele may be detected.
  • the invention also includes nucleic acid molecules, preferably DNA molecules, which are amplified using the polymerase chain reaction under conditions described in Section 5.1.1., infra, and that encode a gene product functionally equivalent to a SCR gene product encoded by any one of the genes and sequences listed in Table 1 or as contained in any one of the clones described herein and deposited with the ATCC.
  • nucleic acid molecules preferably DNA molecules, which are amplified using the polymerase chain reaction under conditions described in Section 5.1.1., infra, and that encode a gene product functionally equivalent to a SCR gene product encoded by any one of the genes and sequences listed in Table 1 or as contained in any one of the clones described herein and deposited with the ATCC.
  • the invention also encompasses (a) DNA vectors that contain any of the foregoing gene and/or coding sequences and/or their complements (i.e., antisense or ribozyme molecules) ; (b) DNA expression vectors that contain any of the foregoing gene and/or coding sequences operatively associated with a regulatory element that directs the expression of the gene and/or coding sequences; and (c) genetically engineered host cells that contain any of the foregoing gene and/or coding sequences operatively associated with a regulatory element that directs the expression of the gene and/or coding sequences in the host cell.
  • regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.
  • the invention also encompasses nucleotide sequences that encode mutant SCJ? gene products, peptide fragments of the SCJ? gene product, truncated SCJ? gene products, and SCR fusion proteins.
  • gene products include, but are not limited to, nucleotide sequences encoding mutant SCR gene products; polypeptides or peptides corresponding to one or more of the Motifs I-VI as shown in FIGS. 13A-F and FIGS.
  • Nucleotides encoding fusion proteins may include but are not limited to full length SCR, truncated SCR or peptide fragments of SCR fused to an unrelated protein or peptide, such as for example, an enzyme, fluorescent protein, or luminescent protein which can be used as a marker.
  • the invention includes, for example, fragments of SCR genes encoding one or more of the following domains as shown in FIG. 5E: amino acids 1-264, 265-283, 287- 316, 410-473, 436-473, and 473-653.
  • homologous SCJ? genes may be identified and may be readily isolated, without undue experimentation, by molecular biological techniques well known in the art. More specifically, such homologs include, for example, paralogs (i.e., members of the SCR gene family occurring in the same plant) as well as orthologs (i.e., members of the SCR gene family which occur in a different plant species) of the Arabidopsis SCR gene.
  • paralogs i.e., members of the SCR gene family occurring in the same plant
  • orthologs i.e., members of the SCR gene family which occur in a different plant species
  • a specific embodiment of a SCR gene and coding sequence of the invention is Arabidopsis SCR (FIGS. 5A and 5E) .
  • Other specific embodiments include the various SCR genes and coding sequences listed in Table 1, supra . Methods for isolating SCR genes and coding sequences are described in detail in Section 5.2, below.
  • SCR genes share substantial amino acid sequence similarities at the protein level and nucleotide sequence similarities in their encoding genes.
  • the term "substantially similar” or “substantial similarity" when used herein with respect to two amino acid sequences means that the two sequences have at least 75% identical residues, preferably at least 85% identical residues and most preferably at least 95% identical residues.
  • the same term when used herein with respect to two nucleotide sequences means that the two sequences have at least 70% identical residues, preferably at least 85% identical residues and most preferably at least 95% identical residues. Determining whether two sequences are substantially similar may be carried out using any methodologies known to one skilled in the art, preferably using computer assisted analysis. For example, the alignments showed herein were initially accomplished by a BLAST search (NCBI using the BLAST network server) . The final alignments of SCJ? family members were done manually. Moreover, SCR genes show highly localized expression in embryos and, particularly, roots. Such expression patterns may be ascertained by Northern hybridizations and in situ hybridizations using antisense probes.
  • the following methods can be used to obtain SCJ? genes and coding sequences from a wide variety of plants, including but not limited to Arabidopsis thaliana , Zea mays , Nicotiana tabacum , Daucus carota , Oryza , Glycine max, Lemna gibba , and Picea abies .
  • Nucleotide sequences encoding an SCR gene or a portion thereof may be obtained by PCR amplification of plant genomic DNA or cDNA.
  • Useful cDNA sources include "free" cDNA preparations (i.e., the products of cDNA synthesis) and cloned cDNA in cDNA libraries. Root cDNA preparations or libraries are particularly preferred.
  • the amplification may use, as the 5'-primer (i.e., forward primer) , a degenerate oligonucleotide that corresponds to a segment of a known SCR amino acid sequence, preferably from the amino-terminal region.
  • the 3'-primer i.e., reverse primer
  • the amino acid sequence of the Arabidopsis SCR protein (SEQ ID NO:2) may be used to design useful 5' and 3' primers.
  • the primers corresponds to segments in the Motif III or VHIID domain of SCR protein (see FIGS. 13B-D and FIGS. 15K-L) .
  • the sequence of the optimal degenerate oligonucleotide probe corresponding to a known amino acid sequence may be determined by standard algorithms known in the art. See for example, Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Vol 2 (1989).
  • the 3'-primer may be an oligonucleotide comprising an 3' oligo(dT) sequence.
  • the amplification may also use as primers nucleotide sequences of SCJ? genes or coding sequences (e .g. , any one of the scr sequences and EST sequences listed in Table 1) .
  • PCR amplification can be carried out, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp") .
  • a Perkin-Elmer Cetus thermal cycler and Taq polymerase Gene Amp
  • stringency of hybridization conditions used in priming the PCR reactions to allow for greater or lesser degrees of nucleotide sequence similarity between the degenerate primers and the corresponding sequences in the cDNA library.
  • the appropriate amplification conditions and parameters depend, in part, on the length and base composition of the primers and that such conditions may be determined using standard formulae.
  • Protocols for executing all PCR procedures discussed herein are well known to those skilled in the art, and may be found in references such as Gelfand, 1989, PCR Technology. Principles and Applications for DNA Amplification. H.A. Erlich, ed. , Stockton Press, New York; and Current Protocols In Molecular Biology. Vol. 2, Ch. 15, Ausubel et al., eds 1988, New York, Wiley & Sons, Inc.
  • a PCR amplified sequence may be molecularly cloned and sequenced.
  • the amplified sequence may utilized as a probe to isolate genomic or cDNA clones of a SCR gene, as described below. This, in turn, will permit the determination of a SCJ? gene's complete nucleotide sequence, including its promoter, the analysis of its expression, and the production of its encoded protein, as described infra .
  • PCR amplification of SCR gene and/or coding sequences can be carried out according to the following procedure: PRIMERS:
  • A.A. code HFTANQAI DNA Sequence: 5' CAT/C TTT/C ACI GCI AAT/C CAA/G GCN AT 3'
  • A.A. code PGGPP(H/N/K) (V/L/F)R' DNA Sequence: 5' CG/T CCA/C GTG/T TGG IGG ICC NCC NGG 3'
  • Useful primer combinations include the following: SCR5AII+SCR3AII; SCR5B+SCR3AII; IF+IR; and IF+4R
  • a SCR gene coding sequence may also be isolated by screening a plant genomic or cDNA library using a SCR nucleotide sequence (e.g., the sequence of any of the SCR genes and sequences and EST clone sequences listed in Table 1.) as hybridization probe.
  • a SCR nucleotide sequence e.g., the sequence of any of the SCR genes and sequences and EST clone sequences listed in Table 1.
  • hybridization probe For example, the whole or a segment of the Arabidopsis SCJ? nucleotide sequence (FIG. 5A) may be used.
  • a SCJ? gene may be isolated from such libraries using as probe a degenerate oligonucleotide that corresponds to a segment of a SCR amino acid sequence.
  • degenerate oligonucleotide probe corresponding to a segment of the Arabidopsis SCR amino acid sequence FIG.
  • RNA is isolated from plant tissues, preferably roots.
  • Poly(A)+ RNA is isolated from the total RNA, and cDNA prepared from the poly(A)+ RNA, all using standard procedures. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual. 2d ed., Vol. 2 (1989).
  • the cDNAs may be synthesized with a restriction enzyme site at their 3'-ends by using an appropriate primer and further have linkers or adaptors attached at their 5'-ends to facilitate the insertion of the cDNAs into suitable cDNA cloning vectors.
  • adaptors or linkers may be attached to the cDNAs after the completion of cDNA synthesis.
  • plant DNA is isolated and fragments are generated, some of which will encode parts of the whole SCR protein.
  • the DNA may be cleaved at specific sites using various restriction enzymes.
  • DNase in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication.
  • the DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis, column chromatography and sucrose gradient centrifugation.
  • the genomic DNA or cDNA fragments can be inserted into suitable vectors, including but not limited to, plasmids, cosmids, bacteriophages lambda or T 4 , and yeast artificial chromosome (YAC) [See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual. 2d ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); Glover, D.M(ed.), DNA Cloning: A Practical Approach. MRL Press, Ltd., Oxford, U.K., Vols. I and II (1985)].
  • suitable vectors including but not limited to, plasmids, cosmids, bacteriophages lambda or T 4 , and yeast artificial chromosome (YAC) [See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual. 2d ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); Glover, D.M(ed.),
  • nucleotide probe DNA or RNA
  • the SCJ? nucleotide probe should be at least 17 nucleotides, preferably at least 26 nucleotides, and most preferably at least 50 nucleotides in length.
  • the nucleotide probe is hybridized under moderate stringency conditions and washed under moderate, preferably high stringency conditions. Clones in libraries with insert DNA having substantial homology to the SCR probe will hybridize to the probe. Hybridization of the nucleotide probe to genomic or cDNA libraries is carried out using methods known in the art. One of ordinary skill in the art will know that the appropriate hybridization and wash conditions depend on the length and base composition of the probe and that such conditions may be determined using standard formulae. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Vol. 2, (1989) pp 11.45-11.57 and 15.55- 15.57.
  • a SCR gene or coding sequence encodes a protein or polypeptide whose amino acid sequence is substantially similar to that of a SCR protein or polypeptide (e.g., the amino acid sequence of any one of the SCR proteins and/or polypeptides shown in FIG. 5A, 5E, FIG. 8, FIG. 9, FIGS. 11A-B, FIGS. 15A-S, FIG. 17B and FIG. 18).
  • the identity of the cloned or amplified SCR gene sequence may be further verified by examining its expression pattern, which should show highly localized expression in the embryo and/or root of the plant from which the SCR gene sequence was isolated.
  • Comparison of the amino acid sequences encoded by a cloned or amplified sequence may reveal that it does not contain the entire SCJ? gene or its promoter.
  • the cloned or amplified SCR gene sequence may be used as a probe to screen a genomic library for clones having inserts that overlap the cloned or amplified SCR gene sequence.
  • a complete SCR gene and its promoter may be reconstructed by splicing the overlapping SCJ? gene sequences.
  • SCR proteins, polypeptides and peptide fragments, mutated, truncated or deleted forms of SCR and/or SCR fusion proteins can be prepared for a variety of uses, including but not limited to the generation of antibodies, as reagents in assays, the identification of other cellular gene products involved in regulation of root development; etc.
  • SCR translational products include, but are not limited to those proteins and polypeptides encoded by the SCR gene sequences described in Section 5.1, above.
  • the invention encompasses proteins that are functionally equivalent to the SCR gene products described in Section 5.1.
  • Such a SCR gene product may contain one or more deletions, additions or substitutions of SCR amino acid residues within the amino adid sequence encoded by any one of the SCJ?
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylaianine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Functionally equivalent”, as utilized herein, refers to a protein capable of exhibiting a substantially similar in vivo activity as the endogenous SCJ?
  • “functionally equivalent” may refer to peptides capable of regulating gene expression in a manner substantially similar to the way in which the corresponding portion of the endogenous SCR gene product would.
  • the invention also encompasses mutant SCR proteins and polypeptides that agree not functionally equivalent to the gene products described in Section 5.1.
  • Such a mutant SCR protein or polypeptide may contain one or more deletions, additions or substitutions of SCR amino acid residues within the amino acid sequence encoded by any one the SCR gene sequences described above in Section 5.1., and which result in loss of one or more functions of the SCR protein (e.g., recognition of a specific nucleic sequence, binding of an transcription factor, etc.), thus producing a SCJ? gene product not functionally equivalent to the wild-type SCR • protein.
  • SCR polypeptides and peptides can be chemically synthesized (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co. , N.Y.) large polypeptides derived from SCR and the full length SCR may advantageously be produced by recombinant DNA technology using techniques well known to those skilled in the art for expressing nucleic acid sequences.
  • RNA capable of encoding SCR protein sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M.J. ed. , IRL Press, Oxford.
  • host-expression vector systems may be utilized to express the SCR gene products of the invention.
  • Such host-expression systems represent vehicles by which the SCJ? gene products of interest may be produced and subsequently recovered and/or purified from the culture or plant (using purification methods well known to those skilled in the art) , but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the SCR protein of the invention in situ .
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli , B .
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing SCR protein coding sequences; yeast (e.g., Saccharomyces , Pichia ) transformed with recombinant yeast expression vectors containing the SCR protein coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the SCR protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing SCJ?
  • yeast e.g., Saccharomyces , Pichia
  • insect cell systems infected with recombinant virus expression vectors e.g., baculovirus
  • plant cell systems infected with recombinant virus expression vectors
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter; the cytomegalovirus promoter/enhancer; etc.) .
  • promoters derived from the genome of mammalian cells
  • mammalian viruses e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter; the cytomegalovirus promoter/enhancer; etc.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the SCR protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not 5 limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with gluta ⁇ thione S-transferase (GST) .
  • GST gluta ⁇ thione S-transferase
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene protein can be released from the GST moiety.
  • full length cDNA sequences are appended with in-frame Bam HI sites at the amino terminus and Eco RI sites at the carboxyl terminus using standard PCR methodologies (Innis et al. , 1990, supra) and ligated into the pGEX-2TK vector (Pharmacia,
  • the resulting cDNA construct contains a kinase recognition site at the amino terminus for radioactive labelling and glutathione S-transferase sequences at the carboxyl terminus for affinity purification (Nilsson, et al., 1985, EMBO J. 4: 1075; Zabeau and Stanley, 1982, EMBO J. 1:
  • the recombinant constructs of the present invention may include a selectable marker for propagation of the construct.
  • a construct to be propagated in bacteria preferably contains an antibiotic resistance gene,
  • Suitable vectors for propagating the construct include plasmids, cosmids, bacteriophages or viruses, to name but a few.
  • the recombinant constructs may include plant-expressible, selectable, or screenable marker genes for isolating, identifying or tracking plant cells transformed by these constructs.
  • Selectable markers include, but are not limited to, genes that confer antibiotic resistance, (e.g., resistance to kanamycin or hygromycin) or herbicide resistance (e.g., resistance to sulfonylurea, phosphinothricin, or glyphosate) .
  • screenable markers include, but are not be limited to, genes encoding ⁇ - glucuronidase (Jefferson, 1987, Plant Mol. Biol. Rep.
  • the recombinant constructs may additionally comprise at least the right T-DNA border sequences flanking the DNA sequences to be transformed into the plant cell.
  • the recombinant constructs may comprise the right and left T-DNA border sequences flanking the DNA sequence.
  • T-DNA based transformation vectors are well known to those skilled in the art.
  • Antibodies that specifically recognize one or more epitopes of SCR, or epitopes of conserved variants of SCR, or peptide fragments of the SCR are also encompassed by the invention.
  • Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs) , humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • various host animals may be immunized by injection with the SCR protein, an SCR peptide (e.g., one corresponding to a functional domain of the protein) , a truncated SCR polypeptide (SCR in which one or more domains has been deleted) , functional equivalents of the SCR protein, or mutants of the SCR protein.
  • SCR proteins, polypeptides, peptides or fusion proteins can be prepared and obtained as described in Section 5.1.2. supra .
  • Host animals may include but are not limited to rabbits, mice, and rats, to name but a few.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum .
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (Nature 256:495-497 [1975]; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al. , 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo . Production of high titers of mAbs in vivo makes this the presently preferred method of production. In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, referred to as complementarily determining regions (CDRs) .
  • CDRs complementarily determining regions
  • humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and a framework region from a human immunoglobulin molecule.
  • techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies against SCR proteins or polypeptides.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • Antibodies to a SCR protein and/or polypeptide can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" SCR, using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, 1993, FASEB J 7(5) :437-444; and Nissinoff, 1991, J. Immunol. 147(8) :2429-2438) .
  • nucleotide sequences including EST clone sequences described in ⁇ 5.1 and 5.1.1. and/or listed in Table 1, and/or polypeptides and proteins described in ⁇ 5.1.2. and/or listed in Table 1, can be used as markers for qualitative trait loci in breeding programs for crop plants.
  • nucleic acid molecules including but not limited to full length SCR coding sequences, and/or partial sequences (ESTs)
  • ESTs partial sequences
  • the SCR gene may encode a product responsible for a qualitative trait that is desirable in a crop breeding program.
  • the SCR protein, peptides and/or antibodies can be used as reagents in immunoassays to detect expression of the SCR gene in cultivars and wild-type plants.
  • SCJ? promoters and functional portions thereof described herein refer to regions of the SCR gene which are capable of promoting tissue-specific expression in embryos and/or roots of an operably linked coding sequence in plants.
  • the SCR promoter described herein refers to the regulatory elements of SCR genes, i .e . , regulatory regions of genes which are capable of selectively hybridizing to the nucleic acids described in Section 5.1, or regulatory sequences contained, for example, in the region between the translational start site of the Arabidopsis SCJ? gene and the Hindlll site approximately 2.5 kb upstream of the site in plasmid pLIGl-3/SAC+Mob21SAC (see FIGS.
  • homologous nucleotide sequences refer to nucleotide sequences including, but not limited to, SCR promoters in diverse plant species (e.g., promoters of orthologs of Arabidopsis SCR) as well as genetically engineered derivatives of the promoters described herein.
  • SCR promoter sequences or portions thereof described herein may be obtained from appropriate plant or mammalian sources from cell lines or recombinant DNA constructs containing SCR promoter sequences, and/or by chemical synthetic methods. SCR promoter sequences can be obtained from genomic clones containing sequences 5' upstream of SCJ? coding sequences.
  • Such 5' upstream clones may be obtained by screening genomic libraries using SCR protein coding sequences, particularly those encoding SCR N-terminal sequences, from SCR gene clones obtained as described in Sections 5.1. and 5.2. Standard methods that may used in such screening include, for example, the method set forth in Benton & Davis, 1977, Science 196:180 for bacteriophage libraries; and Grunstein & Hogness, 1975, Proc. Nat. Acad. Sci. U.S.A. 72:3961-3965 for plasmid libraries.
  • the full extent and location of SCR promoters within such 5' upstream clones may be determined by the functional assay described below.
  • the insert DNA of the clone may be used to isolate genomic clones containing sequences further 5' upstream of the SCR coding sequences.
  • Such further upstream sequences can be spliced on to existing 5' upstream sequences and the reconstructed 5' upstream region tested for functionality as a SCR promoter (i.e., promoting tissue- specific expression in embryos and/or roots of an operably linked gene in plants) . This process may be repeat until the complete SCR promoter is obtained.
  • the location of the SCJ? promoter within genomic sequences 5' upstream of the SCR gene isolated as described above may be determined using any method known in the art.
  • the 3'-end of the promoter may be identified by locating the transcription initiation site, which may be determined by methods such as RNase protection (e.g., Liang et al., 1989, J. Biol. Chem. 264:14486-14498), primer extension (e.g., Weissenborn & Larson, 1992, J. Biol. Chem. 267:6122-6131), and/or reverse transcriptase/PCR.
  • the location of the 3'-end of the promoter may be confirmed by sequencing and computer analysis, examining for the canonical AGGA or TATA boxes of promoters that are typically 50-60 base pairs (bp) and 25-35 bp 5'-upstream of the transcription initiation site.
  • the 5'-end promoter may be defined by deleting sequences from the 5'-end of the promoter containing fragment, constructing a transcriptional or translational fusion of the resected fragment and a reporter gene, and examining the expression characteristics of the chimeric gene in transgenic plants. Reporter genes that may be used to such ends include, but are not limited to, GUS, CAT, luciferase, /3-galactosidase and Cl and R gene controlling anthocyanin production.
  • a SCR promoter is one that confers to an operably linked gene in a transgenic plant tissue-specific expression in roots, root nodules, stems and/or embryos.
  • a SCJ? promoter comprises the region between about -5,000 bp and +1 bp upstream of the transcription initiation site of SCR gene.
  • the Arabidopsis SCJ? promoter comprises the region between positions -2.5 kb and +1 in the 5' upstream region of the Arabidopsis SCR gene (see FIGS. 5A and 14).
  • the cis- regulatory elements within a SCR promoter may be identified using any method known in the art.
  • the location of cis-regulatory elements within an inducible promoter may be identified using methods such as DNase or chemical footprinting (e.g., Meier et al., 1991, Plant Cell 3:309-315) or gel retardation (e.g., Weissenborn & Larson, 1992, J. Biol. Chem. 267-6122-6131; Beato, 1989, Cell 56:335-344; Johnson et al., 1989, Ann. Rev. Biochem. 58:799-839).
  • an inducible promoter-containing fragment may be resected from either the 5' or 3'-end using restriction enzyme or exonuclease digests.
  • the 5'- or 3'-resected fragments, internal fragments to the inducible promoter containing sequence, or inducible promoter fragments containing sequences identified by footprinting or gel retardation experiments may be fused to the 5'-end of a truncated plant promoter, and the activity of the chimeric promoter in transgenic plant examined.
  • Useful truncated promoters to these ends comprise sequences starting at or about the transcription initiation site and extending to no more than 150 bp 5' upstream. These truncated promoters generally are inactive or are only minimally active.
  • truncated plant promoters may include, among others, a "minimal" CaMV 35S promoter whose 5' end terminates at position -46 bp with respect to the transcription initiation site (Skriver et al. , Proc. Natl. Acad. Sci. USA 88:7266-7270); the truncated "-90 35S" promoter in the X-GUS-90 vector (Benfey & Chua, 1989, Science 244:174-181); a truncated "-101 nos” promoter derived from the nopaline synthase promoter (Aryan et al., 1991, Mol. Gen. Genet. 225:65-71); and the truncated maize Adh-1 promoter in pADcat 2 (Ellis et al. , 1987, EMBO J. 6:11-16).
  • a "minimal" CaMV 35S promoter whose 5' end terminates at position -46 bp with respect
  • a cis- regulatory element of a SCR promoter is a sequence that confers to a truncated promoter tissue-specific expression in embryos, stems, root nodules and/or roots.
  • SCR PROMOTER-DRIVEN EXPRESSION VECTORS The properties of the nucleic acid sequences are varied as are the genetic structures of various potential host plant cells. In the preferred embodiments of the present invention, described herein, a number of features which an artisan may recognize as not being absolutely essential, but clearly advantageous are used. These include methods of isolation, synthesis or construction of gene constructs, the manipulation of the gene constructs to be introduced into plant cells, certain features of the gene constructs, and certain features of the vectors associated with the gene constructs.
  • the gene constructs of the present invention may be encoded on DNA or RNA molecules.
  • the desired, stable genotypic change of the target plant be effected through genomic integration of exogenously introduced nucleic acid construct(s) , particularly recombinant DNA constructs.
  • such genotypic changes can also be effected by the introduction of episomes (DNA or RNA) that can replicate autonomously and that are somatically and germinally stable.
  • the introduced nucleic acid constructs comprise RNA
  • plant transformation or gene expression from such constructs may proceed through a DNA intermediate produced by reverse transcription.
  • the present invention provides for use of recombinant DNA constructs which contain tissue-specific and developmental-specific promoter fragments and functional portions thereof.
  • a functional portion of a SCR promoter is capable of functioning as a tissue-specific promoter in the embryo, stem, root nodule and/or root of a plant.
  • the functionality of such sequences can be readily established by any method known in the art. Such methods include, for example, constructing expression vectors with such sequences and determining whether they confer tissue- specific expression in the embryo, stem, root nodule and/or root to an operably linked gene.
  • the invention provides for the use of the Arabidopsis SCR promoter contained in the sequences depicted in FIGS. 5A and 14 and the insert DNA of plasmid pGEX-2TK + .
  • the SCR promoters of the invention may be used to direct the expression of any desired protein, or to direct the expression of a RNA product, including, but not limited to, an "antisense" RNA or ribozyme.
  • RNA product including, but not limited to, an "antisense" RNA or ribozyme.
  • recombinant constructs generally comprise a native SCR promoter or a recombinant SCJ? promoter derived therefrom, ligated to the nucleic acid sequence encoding a desired heterologous gene product.
  • a recombinant SCJ? promoter is used herein to refer to a promoter that comprises a functional portion of a native SCR promoter or a promoter that contains native promoter sequences that is modified by a regulatory element from a SCR
  • a recombinant inducible promoter derived from the scr promoter may be a chimeric promoter, comprising a full-length or truncated plant promoter modified by the attachment of one or more SCR cis-regulatory elements.
  • the manner of chimeric promoter constructions may be any well known in the art. For examples of approaches that can be used in such constructions, see Section 5.1.2., above and Fluhr et al., 1986, Science 232:1106-1112; Ellis et al., 1987, EMBO J. 6:11-16; Strittmatter & Chua, 1987, Proc. Natl. Acad. Sci.
  • the DNA construct is designed so that the protein coding sequence is ligated in phase with the translational initiation codon downstream of the promoter.
  • the promoter fragment is missing 5'leader sequences
  • a DNA fragment encoding both the protein and its 5' RNA leader sequence is ligated immediately downstream of the transcription initiation site.
  • an unrelated 5' RNA leader sequence may be used to bridge the promoter and the protein coding sequence.
  • the design should be such that the protein coding sequence is ligated in phase with the initiation codon present in the leader sequence, or ligated such that no initiation codon is interposed between the transcription initiation site and the first methionine codon of the protein.
  • additional DNA sequences include, but are not limited to, those encoding: a 3' untranslated region; a transcription termination and polyadenylation signal; an intron; a signal peptide (which facilitates the secretion of the protein) ; or a transit peptide (which targets the protein to a particular cellular compartment such as the nucleus, chloroplast, mitochondria, or vacuole) .
  • additional DNA sequences include, but are not limited to, those encoding: a 3' untranslated region; a transcription termination and polyadenylation signal; an intron; a signal peptide (which facilitates the secretion of the protein) ; or a transit peptide (which targets the protein to a particular cellular compartment such as the nucleus, chloroplast, mitochondria, or vacuole) .
  • a desirable plant or plant cell may be obtained by transforming a plant cell with the nucleic acid constructs described herein.
  • Such engineering may be accomplished by transforming a plant or plant cell with all of the desired gene constructs 0 simultaneously.
  • the engineering may be carried out sequentially. That is, transforming with one gene construct, obtaining the desired transformant after selection and screening, transforming the transformant with a second gene construct, and so on.
  • Agrobacterium is employed to introduce the gene constructs into plants.
  • Such transformations preferably use binary Agrobacterium T-DNA vectors (Bevan, 1984, Nuc. Acid Res.
  • the Agrobacterium transformation system may also be 5 used to transform, as well as transfer, DNA to monocotyledonous plants and plant cells (see Hernalsteen et al., 1984, EMBO J 3:3039-3041; Hooykass-Van Slogteren et al., 1984, Nature 311:763-764; Grimsley et al., 1987, Nature 325:1677-179; Boulton et al., 1989, Plant Mol. Biol. 12:31-
  • various alternative methods for introducing recombinant nucleic acid constructs into plants and plant cells may also be utilized. These other methods are particularly useful where the target is a
  • Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al., 1990, Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al., 1988, Proc. Natl. Acad. Sci. USA 85:4305-4309; Gordon-Kamm et al. , 1990, Plant Cell 2:603-618).
  • target plants for engineering include, but are not limited to, crop plants such as maize, wheat, rice, soybean, tomato, tobacco, carrots, peanut, potato, sugar beets, sunflower, yam, Arabidopsis, rape seed, and petunia; and trees such as spruce.
  • desired plants and plant cells may be obtained by engineering the gene constructs described herein into a variety of plant cell types, including but not limited to, protoplasts, tissue culture cells, tissue and organ explants, pollen, embryos as well as whole plants.
  • the engineered plant material is selected or screened for transformants (i.e., those that have incorporated or integrated the introduced gene construct(s) ) following the approaches and methods described below.
  • An isolated transformant may then be regenerated into a plant.
  • the engineered plant material may be regenerated into a plant, or plantlet, before subjecting the derived plant, or plantlet, to selection or screening for the marker gene traits. Procedures for regenerating plants from plant cells, tissues or organs, either before or after selecting or screening for marker gene(s) , are well known to those skilled in the art.
  • Physical and biochemical methods may also be used to identify a plant or plant cell transformant containing the gene constructs of the present invention. These methods include but are not limited to: 1) Southern analysis or PCR amplification for detecting and determining the structure of the recombinant DNA insert; 2) Northern blot, S-l RNase protection, primer-extension or reverse transeriptase-PCR amplification for detecting and examining RNA transcripts of the gene constructs; 3) enzymatic assays for detecting enzyme or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis, western blot techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct products are proteins; 5) biochemical measurements of compounds produced as a consequence of the expression of the introduced gene constructs.
  • a plant that expresses a recombinant SCR gene may be engineered by transforming a plant cell with a gene construct comprising a plant promoter operably associated with a sequence encoding SCR protein or a fragment thereof.
  • a plant promoter operably associated is used herein to mean that transcription controlled by the "associated" promoter would produce a functional messenger RNA, whose translation would produce the enzyme.
  • the plant promoter may be constitutive or inducible.
  • ERTAIN promoters include, but are not limited to, the CaMV 35S promoter, the T-DNA mannopine synthetase promoter, and their various derivatives.
  • Useful inducible promoters include but are not limited to the promoters of ribulose bisphosphate carboxylase (RUBISCO) genes, chlorophyll a/b binding protein (CAB) genes, heat shock genes, the defense responsive gene (e.g., phenylaianine ammonia lyase genes), wound induced genes (e.g., hydroxyproline rich cell wall protein genes), chemically-inducible genes (e.g., nitrate reductase genes, gluconase genes, chitinase genes, PR-1 genes etc .
  • RUBISCO ribulose bisphosphate carboxylase
  • CAB chlorophyll a/b binding protein
  • heat shock genes e.g., the defense responsive gene (e.g., phenylai
  • dark-inducible genes e.g., asparagine synthetase gene (Coruzzi and Tsai, U.S. Patent 5,256,558, October 26, 1993, Gene Encoding Plant Asparagine Synthetase) developmentally regulated genes (e.g., Shoot Meristemless gene) to name just a few.
  • 35 structural elements of different promoters have unique expression patterns and/or levels not found in natural promoters. See, e .g . , Salina et al., 1992, Plant Cell 4:1485-1493, for examples of artificial promoters constructed from combining cis-regulatory elements with a promoter core.
  • the associated promoter is a strong and root, root nodule, stem and/or embryo-specific plant promoter such that the SCR protein is overexpressed in the transgenic plant.
  • root- and root nodules-specific promoters include but are not limited to the promoters of SCR genes, SJfJ? genes, legehemoglobin genes, nodulin genes and root-specific glutamine synthetase genes (See e .g. , Tingey et al., 1987, EMBO J. 6:1-9; Edwards et al., 1990, Proc. Nat. Acad. Sci. USA 87:3459-3463) .
  • the overexpression of SCR protein in roots may be engineered by increasing the copy number of the SCR gene.
  • One approach to producing such transgenic plants is to transform with nucleic acid constructs that contain multiple copies of the complete SCR gene (i.e., with its own native scr promoter) .
  • Another approach is repeatedly transform successive generations of a plant line with one or more copies of the complete SCR gene.
  • Yet another approach is to place a complete SCJ? gene in a nucleic acid construct containing an amplification-selectable marker (ASM) gene such as the glutamine synthetase or dihydrofolate reductase gene.
  • ASM amplification-selectable marker
  • a desired plant may be engineered by suppressing SCR activity.
  • the suppression may be engineered by transforming a plant with a gene construct encoding an antisense RNA or ribozyme complementary to a segment or the whole of SCR RNA transcript, including the mature target mRNA.
  • SCR gene suppression may be engineered by transforming a plant cell with a gene construct encoding a ribozyme that cleaves the SCR mRNA transcript.
  • the plant can be engineered, e .g. , via targeted homologous recombination to inactive or "knock-out" expression of the plant's endogenous SCR.
  • suppression constructs express specifically in the root, root nodule, stem and/or embryo tissues.
  • the suppression constructs may be preferred to have the suppression constructs expressed constitutively.
  • constitutive promoters such as the nopaline, CaMV 35S promoter, may also be used to express the suppression constructs.
  • a most preferred promoter for these suppression constructs is a SCJ? or SHR promoter.
  • desired plants with suppressed target gene expression may also be engineered by transforming a plant cell with a co-suppression construct.
  • a co-suppression construct comprises a functional promoter operatively associated with a complete or partial SCJ? gene sequence. It is preferred that the operatively associated promoter be a strong, constitutive promoter, such as the CaMV 35S promoter. Alternatively, the co-suppression construct promoter can be one that expresses with the same tissue and developmental specificity as the scr gene.
  • the co-suppression construct encodes a incomplete SCR mRNA, although a construct encoding a fully functional SCR mRNA or enzyme may also be useful in effecting co- suppression.
  • desired plants with suppressed target gene expression may also be engineered by transforming a plant cell with a construct that can effect site-directed mutagenesis of the SCR gene.
  • a construct that can effect site-directed mutagenesis of the SCR gene See, e.g., Offringa et al., 1990, EMBO J. 9:3077-84; and Kanevskii et al., 1990, Dokl. Akad. Nauk. SSSR 312:1505-1507) for discussions of nucleic constructs for effecting site-directed mutagenesis of target genes in plants.
  • constructs effect suppression of SCJ? gene by replacing the endogenous SCR gene sequence through homologous recombination with none or inactive SCR protein coding sequence.
  • a desired plant may be engineered to express a gene of interest under the control of the SCJ? promoter.
  • SCJ? promoters and functional portions thereof refer to regions of the nucleic acid sequence which are capable of promoting tissue-specific transcription of an operably linked gene of interest in the embryo, stem, root nodule and/or root of a plant.
  • the SCR promoter described herein refers to the regulatory elements of SCJ? genes as described in Section 5.2.
  • Genes that may be beneficially expressed in the roots and/or root nodules of plants include genes involved in nitrogen fixation or cytokines or auxins, or genes which regulate growth, or growth of roots.
  • genes encoding proteins that confer on plants herbicide, salt, or pest resistance may bt engineered for root specific expression.
  • the nutritional value of root crops may also be enhanced through SCJ? promoter driven expression of nutritional proteins.
  • therapeutically useful proteins may be expressed specifically in root crops.
  • Genes that may be beneficially expressed in the stems of plants include those involved in starch lignin or cellulose biosynthesis. _
  • desired plants which express a heterologous gene of interest under the control of the SCR promoter may be engineered by transforming a plant cell with SCR promoter driven constructs using those techniques described in Section 5.2.2. and 5.3., supra .
  • transgenic plants having the desired engineered traits screening of transformed plants (i.e., those having an gene construct of the invention) having those traits may be required.
  • transformed plants are examined for those expressing the SCR gene at the desired level and in the desired tissues and developmental stages.
  • SCJ? gene product e.g., RNA or protein
  • overexpression or SCR suppression may then be subsequently screened for those plants that have the desired structural changes at the plant level (e.g., transgenic plants with overexpression or suppression of SCR gene having the desired altered root structure) .
  • the same principle applies to obtaining transgenic plants having tissue-specific expression of a heterologous gene in embryos and/or roots by the use of a SCJ? promoter driven expression construct.
  • the transformed plants may be directly screened for those exhibiting the desired structural and functional changes.
  • such screening may be for the size, length or pattern of the root of the transformed plants.
  • the screening of the transformed plants may be for altered gravitropism or decreased susceptibility to lodging.
  • the screening of the transformed plants may be for improved agronomic characteristics (e.g., faster growth, greater vegetative or reproductive yields, or improved protein contents, etc.), as compared to unengineered progenitor plants, when cultivated under various growth conditions (e.g., soils or media containing different amount of nutrients, water content) .
  • plants engineered with SCR overexpression may exhibit improved vigorous growth characteristics when cultivated under conditions where large and thicker roots are advantageous.
  • Plants engineered for SCJ? suppression may exhibit improved vigorous growth characteristics when cultivated under conditions where thinner roots are advantageous.
  • Engineered plants and plant lines possessing such improved agronomic characteristics may be identified by examining any of following parameters: 1) the rate of growth, measured in terms of rate of increase in fresh or dry weight; 2) vegetative yield of the mature plant, in terms of fresh or dry weight; 3) the seed or fruit yield; 4) the seed or fruit weight; 5) the total nitrogen content of the plant; 6) the total nitrogen content of the fruit or seed; 7) the free amino acid content of the plant; 8) the free amino acid content of the fruit or seed; 9) the total protein content of the plant; and 10) the total protein content of the fruit or seed.
  • the procedures and methods for examining these parameters are well known to those skilled in the art.
  • a desired plant is one that exhibits improvement over the control plant (i.e., progenitor plant) in one or more of the aforementioned parameters.
  • a desired plant is one that shows at least 5% increase over the control plant in at least one parameter.
  • a desired plant is one that shows at least 20% increase over the control plant in at least one parameter.
  • Most preferred is a plant that shows at least 50% increase in at least one parameter.
  • EXAMPLE 1 ARABIDOPSIS SCR GENE This example describes the cloning and structure of the Arabidopsis SCR gene and its expression.
  • the deduced amino acid sequence of the Arabidopsis SCR gene product contains a number of potential functional domains similar to those found in transcription factors. Closely related sequences have been found in both dicots and monocots indicating that Arabidopsis SCR is a member of a new protein family.
  • the expression pattern of the SCR gene was characterized by means of in situ hybridization and by an enhancer trap insertion upstream of the SCR gene (described in more detail in Section 7) .
  • the expression pattern is consistent with a key role for Arabidopsis SCR in regulating the asymmetric division of the cortex/endodermis initial which is essential for generating the radial organization of the root.
  • a RFLP for the SCR gene was identified between Col and Ler ecotypes with Xho I endonuclease. Genomic DNAs from independent Rl lines (Jarvis et al., 1994, Plant Mol. Biol.
  • Morphological characterization of the mutant roots was performed as follows: 7 to 14 days post-germination phenotypically mutant seedlings were fixed in 4.0%
  • genomic clone containing an insert of approximately 23 kb was isolated.
  • Comparison of the nucleotide sequence between the genomic clone and the rescued plasmid revealed the site of the T-DNA insertion.
  • Four cDNA clones were isolated. The dideoxy sequencing method was performed using the Sequenase kit (United States Biochemical Corp.).
  • RNA from plant tissues was obtained using phenol/chloroform extractions as described in (Berry et al., 1985, Mol. Cell. Biol. 5:2238-2246) with minor modifications. Northern hybridization and detection were performed according to the Genius kit manual (Boehringer Mannheim) .
  • genomic DNA from ET199 homozygous plants was amplified using primers specific for the T-DNA left border and the SCR gene. An approximately 2.0 kb fragment was amplified. This fragment was sequenced and the site of insertion was found to be approximately 1 kb from the ATG start codon.
  • Antisense and sense SCR riboprobes were labeled with digoxigenin-ll-UTP (Boehringer Mannheim) using T7 polymerase following the manufacturer's protocol. Probes contained a 1.1 kb 3' portion of the cDNA. Probe purification, hydrolysis and quantification were performed as described in the Boehringer Mannheim Genius System user's guide. Tissue samples were fixed in 4 % formaldehyde overnight at 4°C and rinsed two times in PBS (Jackson et al., 1991, PI. Cell 3:115-125). They were subsequently pre- embedded in 1 % agarose in PBS. The fixed tissue was dehydrated in ethanol, cleared in Hemo-De (Fisher Scientific, Pittsburgh, PA) and embedded in ParaplastPlus (Fisher
  • Tissue sections (lO ⁇ m thick) were mounted on SuperfrostPlus slides (Fisher Scientific) . Section pretreatment and hybridization were performed according to (Lincoln et al., 1994, Plant Cell 6:1859-1876) except that proteinase K was used at 30 mg/ml and a two hour prehybridization step was included. Probe concentration of 50 ng/ml/kb was used in the hybridization.
  • Slides were washed and the immunological detection was performed according to (Coen et al., 1990, Cell 63:1311- 1322) with the following modifications. Slides were first washed 5 h in 5xSSC, 50% formamide. After RNase treatment slides were rinsed three times (20 min each) in the buffer (0.5 M NaCl, 10 mM Tris-HCI pH 8.0, 5.0 mM EDTA). In the immunological detection, antibody was diluted 1:1000, levamisole (240 ng/ml) was included in the detection buffer, and after stopping the reaction in 10 mM Tris, 1 mM EDTA, sections were mounted directly to Aqua-Poly/Mount (Polysciences, Warrington, PA) .
  • the F2 progeny of this cross were all mutant and segregated 3:1 for antibiotic resistance confirming allelism (see Materials & Methods) .
  • the principal phenotypic difference between the two alleles was that scr-1 root growth was more retarded than that of scr-2 , suggesting that it is the stronger allele (FIG. 2A) .
  • the aerial organs appeared similar to wild-type and the flowers were fertile (FIGS. 2A and 2B) .
  • the progeny of backcrosses of scr-1 or scr-2 to wild-type plants segregated 3:1 for the root phenotype for both alleles, indicating that each mutation is monogenic and recessive.
  • mutant root closely resembles that of wild-type except for the consistent reduction in the number of cell layers. Because the endodermis and cortex are normally generated by an asymmetric division of the cortex/endodermal initial, this indicates that the primary defect in scr is disruption of this asymmetric division.
  • the cell layer could have differentiated attributes of either cortex or endodermis. Alternatively, it could have an undifferentiated, initial-cell identity or it could have a chimeric identity with differentiated attributes of both endodermis and cortex in the same cell.
  • this histochemical stain also reveals the presence of lignin, indicating the presence of differentiated xylem cells in mutant (FIG. 3A) and wild-type (FIG. 3B) .
  • Another marker of the differentiated endodermis is the arabinogalactan epitope recognized by the monoclonal antibody, JIM13 (Knox et al., 1990, Planta 181:512-521).
  • JIM13 The mutant cell layer showed staining with this antibody (FIG. 3C, compare with wild-type, FIG. 3B) .
  • the JIM7 antibody that recognizes pectin epitopes in all cell walls was used (FIGS. 3E and 3F) .
  • the cell layer between the epidermis and the pericycle has differentiated attributes of the endodermis.
  • the CCRC-M2 monoclonal antibody As a marker for the cortex, the CCRC-M2 monoclonal antibody was used. This antibody recognizes a cell wall oligosaccharide epitope, found only on differentiated cortex and epidermis cells. In sections from the differentiation zone of scr-1 and scr-2 , both cortex and epidermal cells showed staining (FIG. 4A and 4B) that was similar to that of wild-type (FIG. 4C) . In scr-1 , staining of both cell types was apparent, but staining of cortex was somewhat weaker than wild-type. The positive control used the CCRC-M1 monoclonal antibody which recognizes an oligosaccharide epitope found on all cells (FIGS. 4D-F) .
  • the Arabidopsis SCJ? gene product is a 653 amino acid polypeptide that contains several domains (FIG. 5B) .
  • the amino-terminus has homopolymeric stretches of glutamine, serine, threonine, and proline residues, which account for 44% of the first 267 residues. Domains rich in these residues have been shown to activate transcription and may serve such a role in SCR (Johnson et al., 1993, J. Nutr. Biochem 4:386-398).
  • a charged region between residues 265 and 283 has similarity to the basic domain of the bZIP family of transcriptional regulatory proteins (FIG. 5C) (Hurst, H.C., 1994, Protein Profile 1:123-168).
  • the basic domains from several bZIP proteins have been shown to act as nuclear localization signals (Varagona et al., 1992, Plant Cell 4:1213-1227), and this region in SCR may act similarly. This charged region is followed by a leucine heptad repeat (residues 291-322) . A second leucine heptad repeat is found toward the carboxy-terminus (residues 436 to 473) . As leucine heptad repeats have been demonstrated to mediate protein-protein interactions in other proteins (Hurst, H.C., 1994, Protein Profile 1:123-168), the existence of these motifs suggests that SCR may function as a dimer or a multimer.
  • the second leucine heptad repeat is followed by a small region rich in acidic residues, also present in a number of defined transcriptional activation domains (Johnson et al., 1993, J. Nutr Biochem 4:386-398). While each of these domains has been found within proteins that do not act as transcriptional regulators, the fact that all of them are found within the deduced SCR protein sequence indicates that SCR is a transcriptional regulatory protein.
  • the Arabidopsis SCR protein sequence was compared with the sequences in the available databases. Eleven expressed sequence tags (ESTs) , nine from Arabidopsis, one from rice and one from maize, showed significant similarity to residues 394 to 435 of the SCR sequence, a region immediately amino-terminal to the second leucine heptad repeat (FIGS. 15K-L) . This region is designated the VHIID domain. Subsequent analysis of these EST sequences has revealed that the sequence similarity extends beyond this region; in fact, the similarity extends throughout the entire known gene products.
  • ESTs expressed sequence tags
  • RNA blot analysis revealed expression of SCJ? in Arabidopsis siliques, leaves and roots of wild-type plants (FIG. 6A) .
  • No hybridization was detected to RNA from scr-1 plants (FIG. 6B, lane 2) .
  • scr-1 has a reduced level of RNA expression and may represent the null 0 phenotype.
  • Hybridization to RNA species larger than the normal size were detected in s ⁇ r-2. This indicates that abnormal SCJ? transcripts are made in this allele, suggesting that functional but possibly altered proteins may be produced.
  • RNA in situ was hybridization performed on sections of root tissue. In mature roots, expression was localized primarily to the endodermis (FIGS.
  • enhancer trap (ET) lines were prepared and examined in which the ⁇ -glucuronidase (uid-A or GUS) coding sequence with a minimal promoter was expressed in the root endodermis. (See Section 7, infra) . Restriction fragment length polymorphisms were observed when DNA from one of these lines, ET199 and wild-type were probed with SCR. PCR and sequence analysis confirmed that the enhancer-trap construct had inserted approximately 1 kb upstream of the SCR start site and in the same orientation as that of SCR transcription.
  • the formation of the cortex and endodermal layers in the Arabidopsis root requires two asymmetric divisions.
  • an anticlinal division of the cortex/endodermal initial generates two cells with different developmental potentials. One will continue to function as an initial, while the other undergoes a periclinal division to generate the first cells in the endodermal and cortex cell files.
  • This second asymmetric division is eliminated in the scarecrow mutant, resulting in a single cell layer instead of two.
  • the scr mutation appears to have little effect on any other cell divisions in the root indicating that it is involved in regulating a single asymmetric division in this organ.
  • Several other mutations have been characterized that appear to affect specific cell division pathways in Arabidopsis.
  • At least one additional cell division appears to be affected in the scr mutant.
  • the ground tissue does not divide to form the endodermal and cortex layers of the embryonic root and hypocotyl.
  • expression of SCR was detected in the endodermal tissue throughout the embryonic axis shortly after this division occurs.
  • SCJ? may play a direct role in regulating both this division and the division of the cortex/endodermal initial in the root apical meristem.
  • the radial organization established in the embryo may somehow act as a template that directs the division of the cortex/endodermal initial, thus perpetuating the pattern. This is consistent with the finding in the scr mutant that the aberrant pattern established in the embryo i ⁇ perpetuated in the primary root.
  • the cloning of the gene and the expression pattern provide some clues as to the role of SCJ? in the regulation of a specific asymmetric division.
  • the SCR gene is expressed in the cortex/endodermal initial, but immediately after division is restricted to the endodermal lineage.
  • a similar pattern is seen in the ET199 enhancer trap line in which SCJ? regulatory elements are in proximity to a GUS gene, indicating that SCJ? restriction to the endodermal cell file is due to differential regulation of expression of the SCR gene in this cell and the first cell in the cortex file.
  • Another marker line in which expression of GUS is detected only in the cortex daughter cell provides a control for differential degradation of GUS RNA or protein.
  • GenBank database reveals that the proteins they encode share a high degree of homology with Arabidopsis SCR protein. See Table 1 and FIGS. 15A-S. Further sequence analysis of the encoded proteins indicate that a high degree of sequence similarity extends from at least the highly conserved VHIID domain to the carboxy-terminus of the gene products. Comparison of the amino termini of these proteins is precluded by the fact that the ESTs are incomplete. The high degree of similarity among these proteins, in combination with the motifs observed in the SCR protein (homopolymeric motifs, two leucine heptad repeats and a bZIP-like basic domain that may also function as a nuclear localization sequence) indicates that these proteins form a novel class of regulatory proteins.
  • EXAMPLE 2 ENHANCER TRAP ANALYSIS OF ROOT DEVELOPMENT An enhancer trap system was used in order to provide a more detailed molecular analysis of gene expression in lateral root patterning and development in Arabidopsis thaliana .
  • a new collection of marker lines that express ⁇ - glucuronidase (GUS) activity in a cell-type specific manner in each of the cells of the root was generated. These lines allow differentiation of cells to be monitored based on molecular characteristics.
  • GUS ⁇ - glucuronidase
  • ET199 resulted from the integration of the GUS cassette in proximity to an SCR enhancer.
  • the results described below demonstrate that transcriptional activation of the SCR gene plays an important role in root development in Arabidopsis, and that SCR gene transcriptional regulatory elements can express a transgene in a developmentally and tissue specific manner.
  • Roots were removed from plates and infiltrated in 25% glycerol for several hours to overnight. Roots were then mounted in 50% glycerol. Whole seedlings were stained for GUS activity for up to three days in the following solution: IX GUS buffer, 20% methanol, 0.5 mg/ml X-Glu. Addition of methanol greatly improves the specificity and reproducibility of staining. Staining solution was made fresh from a 10X buffer (1 M Tris pH7.5, 290 mg NaCl, 66 mg K 3 Fe(CN) 6 ) that was stored for no more than one week. Stained roots were cleared in glycerol and mounted as above. All samples were observed using Nomarski optics on a Leitz Laborlux s microscope.
  • AgroJbacteriujn mediated root transformation (Marton & Browse, 1991, Plant Cell Reports 10:235-239), and 4 independent lines from each transformant were screened for GUS activity in the root.
  • the marker lines described above reflect patterns of gene expression that are specific to individual root cell types. There are no readily apparent mutant phenotypes in any of these lines. Therefore, they can be used to analyze the differentiation state of the cells during normal development of the lateral root primordial (LRP) . If there are stages at which the pericycle cells proliferate in the absence of patterning, it can be expected that all cells would be identical with none expressing differentiated characteristics. In contrast, organization of the LRP would be reflected in differential patterns of GUS gene expression, with certain cells beginning to turn on transcription from differentiated cell-type specific promoters (i.e., those that drive GUS expression in the enhancer trap lines) .
  • LRP lateral root primordial
  • Stage I The LRP is first visible as a set of pericycle cells that are clearly shorter in length than their neighbors, having undergone a series of anticlinal divisions. Laskowski et al., 1995, Dev. 121:3303-3310 predict that there are approximately 4 founder pericycle cells involved. In the longitudinal plane, these divisions result in the formation of 8-10 small cells, which enlarge in a radial direction.
  • Stage II A periclinal division occurs that divides the LRP into two layers (Upper Layer (UL) and Lower Layer (LL) ) . Not all the small pericycle-derived cells appear to participate in this division — typically the most peripheral cells do not divide. Hence, as the UL and LL cells expand radially the domed shape of the LRP begins to appear.
  • UL Upper Layer
  • LL Lower Layer
  • Stage III The UL divides periclinally, generating a three layer primordium comprised of ULl, UL2 and LL. Again, some peripheral cells do not divide, creating peripheral regions that are one and two cell layers thick. This further emphasizes the domed shape of the LRP.
  • Stage IV The LL divides periclinally, creating a total of four cell layers (ULl, UL2, LL1, LL2) . At this stage the LRP has penetrated the parent endodermal layer.
  • Stage V The central cells in LL2 undergo a number of divisions that push the overlying layers up and distort the cells in LL1. These divisions are difficult to visualize at this stage, but clearly form a knot of mitotic activity.
  • the LRP at this stage is midway through the parent cortex.
  • the outer layer contains 10-12 cells.
  • Stage VI This stage is characterized by several events.
  • the four central cells of ULl divide periclinally. This division is particularly useful in identifying the median longitudinal plane in the enlarging LRP. At this point there are a total of twelve cells in ULl, four in the middle that have undergone the periclinal division and four on either side. In addition, all but the most central cells of UL2 undergo a periclinal.division. At this point the LRP has passed through the parent cortex layer and has penetrated the epidermis. The central cells apparently derived from LL2 have a distinct elongated shape characteristic of vascular elements.
  • Stage VII As the primordium enlarges it becomes difficult to characterize the divisions in the internal layers.
  • the cells in the outermost layer can still be seen very clearly. All of these cells undergo a anticlinal division, resulting in 16 central cells (8 cells in each of two layers) flanked by 8-10 cells on each side. We refer to this as the 8-8-8 cell pattern.
  • the LRP appears to be just about to emerge from the parent root.
  • An enhancer trapping cassette was generated by fusing the GUS coding sequence to the minimal promoter of the 35S promoter from CaMV.
  • This minimal promoter does not produce a detectable level of GUS expression.
  • its presence allows other upstream elements to direct GUS expression in a developmental and/or cell-specific manner (Benfey et al., 1990, EMBO J. 9:1677-1684).
  • the use of a minimal promoter instead of a promoterless construct allows GUS expression to occur even if the enhancer trap cassette inserts at a distance from the coding region. Since the insert does not have to be within the structural gene, there are often no mutations generated in the enhancer trap lines.
  • the minimal promoter:GUS construct was cloned immediately adjacent to the T-DNA right border sequence of PCV (Koncz et al., supra) and introduced into Arabidopsis. 350 independent lines were generated and analyzed for GUS activity in the root. The following lines most clearly define each cell type. All of the lines were generated through enhancer trapping, as described herein, below, except for CorAX92 (Dietrich et al., 1992, Plant Cell 4:1371-1382) and EpiGL2:GUS (Masucci et al., Dev. 122:1253-1260) which are transgenic plants that contain cell-type specific promoters fused to the GUS gene.
  • Endl95 - expresses GUS in the endodermis of primary and lateral roots. Staining can be seen most clearly in the cells in the meristematic region of the root, although overstaining shows that more mature cells also express some GUS activity. It appears that there is no staining in the cortex/endodermal initial, but staining is evident in the first daughter cell of this initial. GUS expression is also seen at the base of young leaves and in the stipules.
  • ET199 expresses GUS in the endodermis of primary and lateral roots, again most clearly in cells in the meristematic region. Unlike Endl95, staining in ET199 appears to continue down to the cortex/endodermal initial and, in younger roots, even into the cells of the quiescent center. Expression in the aerial parts of the plant is detectable in the young leaf primordia.
  • CorAX92 - This line was generated by fusing the 5' and 3' sequences from a cortex specific gene isolated from oilseed rape to the GUS reporter gene (Dietrich et al., Plant Cell 4:1371-1382). Expression is limited to the cortex layer, extending to but not including the cortex/endodermal initial. Staining is also apparent in the petioles and leaf blades of expanded leaves.
  • EpiGL2:GUS - This line was generated by fusing the GL2 promoter to the GUS gene (Masucci et al., Dev. 122:1253- 1260) . Expression is seen in the non-hair forming epidermal cells (atrichoblasts) .
  • Two marker lines show differential staining at very early stages of LRP development.
  • ET199 presents a complex and dynamic pattern of expression. Staining is first apparent at stage II in only the four central cells of the UL. At stage III staining is strongest in the central cells of UL2. As the LRP reaches stage V the staining remains strongest in the central 2-4 cells of UL2. By stage VI staining also begins to extend into the newly formed endodermal layer, and staining in both the central cells and endodermis persists beyond emergence of the lateral root.
  • Another line, LRB10 (lateral root base) does not express GUS in the primary root tip.
  • stage I Staining in the LRP is seen at stage I, and at stage II all the cells of the UL and LL are stained. However, by stage IV and V only the cells at the periphery of the LRP are still expressing GUS. As the LRP develops, these cells continue to stain, although less intensely, resulting in a ring of GUS expressing cells at the base of the LR. LRB10 and ET199 clearly demonstrate non-identity between the cells at very early stages, stage IV in the case of LRB10 and within the UL at stage II in ET199.
  • the expression pattern of Arabidopsis SCJ? has been determined by analysis of an enhancer trap line, ET199, in which a GUS coding region with a minimal promoter was fortuitously inserted 1 kb upstream of the SCJ? coding region
  • GUS expression is detected in the endodermis, endodermal initials and sometimes in the quiescent center (QC) of the root. See supra and Malamy and
  • the expression of the SCR promoter::GUS construct was also examined in scr mutant background.
  • the scr mutant has an altered root organization (see, supra) . Whereas the wild-type root of Arabidopsis has four distinct cell layers surrounding the vascular tissue, the roots of scr mutant have only three.
  • Transgenic roots of the scr mutant were generated that contained a SCJ? promoter::GUS construct. As in the wild-type, a large number of transgenic roots were formed that had detectable GUS expression ( Figure 20, Panel a) . These roots were shorter than wild-type regenerated roots, consistent with the shorter root phenotype of the scr mutant. Additional transgenic root experiments demonstrated that the SCR gene under control of its own promoter can rescue the scr mutant phenotype.
  • Transgenic scr roots were generated that contained the full length SCR gene under the control of its own promoter. The length of transgenic roots containing the construct were longer than those of the scr mutant, indicating that the introduced SCR gene partially rescued the mutant. Whereas scr regenerated roots that carried the SCR promoter::GUS construct were very short ( Figure 21, Panel a; and Figure 20, Panel a) , roots transformed with the SCJ? promoter and coding region were noticeably longer ( Figure 21, Panel b) . The difference was even more obvious in liquid culture, in which scr mutant roots remained short ( Figure 21, Panel c) , while SCR gene complemented scr mutant roots were long and resembled wild- type roots ( Figure 21, Panel d) .
  • degenerate primers SCR3AII, SCR5AII and SCR5B were designed and used in PCR amplification of SCR sequences from genomic DNA of various plant species.
  • the 146 bp amplification product, ZmScll was subsequently used as a probe for screening of a genomic library generated in lambda BlueSTAR vector (NOVAGEN) from maize (Hill line) genomic DNA.
  • the screening was performed according to the standard procedures described in GeniusTM System User's Guide For Membrane Hvbridization (Boehringer- Mannheim) :
  • the probe was a single-strand DNA molecule corresponding to the ZmScll fragment produced by PCR (Genius, Boehringer-Mannheim) .
  • Hybridization was performed according to recommendations of the manufacturer's manual (Boehringer-Mannheim) . Prehybridization was for 2 hr in 50% formamide hybridization solution at 42°C.
  • Hybridization was overnight at 42°C with 200 ng/ml probe concentration. Filters were washed twice at room temperature in 2xSSC, 0.1% SDS for 5 min, and for stringent washing at 65°C in 0.5xSSC,0.1% SDS twice for 15 min.
  • the clone contained a 13 kb insert, which was subcloned into a plasmid vector. The resulting plasmid was designated pZCR.
  • a 5 kb Eco RI fragment containing the maize SCR (ZCR) sequence was subcloned and sequenced.
  • the nucleotide sequence of the region containing a partial ZCR coding sequence is shown in FIG. 17A and the corresponding deduced amino acid sequence is shown in FIG. 17B.
  • the ZCR protein contain a segment that is highly homologous to a corresponding segment in the Arabidopsis SCR protein (FIG. 17B) . This segment is flanked by segments of low homology.
  • the genomic clone of ZCR is a composite clone, containing sequences that are not ZCR sequences.
  • the deduced ZCR protein sequence was aligned with that of Arabidopsis SCR protein.
  • the comparison revealed new conserved sites in the SCJ? coding sequence which were used to design new, more specific PCR primers (i.e., IF, IR, and 4R) for use in amplification of SCR sequences from yet other plant species.
  • PCR amplification were performed as described in section 5.1.1.
  • Two DNA of expected size were obtain from soybean: a 247 bp DNA from the 1F+1R primer combination and a 379 bp DNA from the 1F+4R primer combination.
  • a DNA of expected size (247 kb) was obtained from carrot and spruce when their genomic DNA was amplified using 1F+4R primer combination.
  • the nucleotide sequences of the 379 kb soybean DNA (SRPgl) , the 247 kb DNA from carrot (SRPdl ) and spruce (SRPpl ) are shown in FIGS. 16K-M.
  • RNA probe was subjected to mild alkali hydrolysis by heated at 60°C for 1 hr in 100 mM carbonate buffer (pH 10.2) to yield a probe size of approximately 0.15 kb. Probe concentration for hybridization was optimized at 1 ⁇ g/ml/kb. In situ hybridization of root tips from 48 to 72 hr-old maize seedlings or excised quiescent centers (QCs) of roots were carried out following procedures described in Section 6.1.6. , supra .
  • ZCR expression during regeneration of the root apex was also examined.
  • cell proliferation occurs to fill in the removed tissue and begins to regenerate the basic shape of the root tip. All cells on the blunt edge of the root appears to contribute to the new population of cells.
  • the ZCR expression pattern indicates that molecular signals are differentially present in these cells at an early stage in regeneration.
  • the gene appears to be diagnostic of cells that are preparing to undergo asymmetrical division in order to re-establish the normal organization of the root apex from the large undifferentiated cells.
  • the results indicate that ZCR expression is required for pattern formation since it is expressed prior to the generation of any specific anatomical pattern in the newly formed not tissue.
  • SCR is expressed in the endodermis. Expression was also found in cells at the root tip that are located at the distal end of the endodermal cell files. In soybean nodules, expression of SCR was detected in the peripheral tissue at the site of developing vascular strands. At later stages of vascular development within the nodule, SCR expression was found flanking the vascular tissue. These results indicate that SCR is involved in regulating vascularization in the nodule by contributing to the radial organization that is required to generate endodermis. These findings indicate that SCR promoter may be used to express proteins in a highly tissue-specific manner in soybean nodules. One application is to use SCR promoter to engineer nodules through production of components in a tissue-specific manner.
  • both the scr and the shr mutants have shoots that exhibit severely defective gravitropism.
  • Complementation analysis showed that scr is allelic to a sgr (shoot gravitropism) mutant, sgrl .
  • sgr shoot gravitropism
  • Four mutant alleles of SCR i .e . , scrl , scr2, sgrl-1 and sgr 1-2 have been identified. All four of these mutants have normal root gravitropism and defective shoot gravitropism.
  • Etiolated hypocotyls of scr mutants placed on their sides do not respond to gravity even after 3 hr. Similar behaviors were observed with the inflorescence stems of sgrl-1 mutant, which do not curve upwards even after two days on their sides. In contrast, the roots of these plants respond rapidly to the change in orientation with the same kinetics as the wild type. Thus, mutations in the SCR gene lead to a radial pattern deficiency in the root but have no effect on root gravitropism.
  • MOLECULE TYPE DNA (genomic)
  • Ala lie lie lie Arg Asp Leu lie His Ser Ser Thr Ser Val Ser lie Pro 145 150 155 160
  • ATCCGTGCTC TTGGTGCTAG ACCTGGTGGA CCTCCGAACG TTAGGATAAC GGGAATTGAT 240
  • AAAAA 1085 INFORMATION FOR SEQ ID NO:19:
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:20:
  • ACAAAAGTTC CAAGAACGTC TCAAGATAGG ATCAAAGTGG AGAAGATGCT CTTCGGGGAG 720
  • CTGTCTCTTT ATGCTATTTT GGCTTAAATG CTTCTACTGC CTCTGCATGT AAAGCCTTTG 1140
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:22:
  • AAAAAAAAAA AAAA 1094 INFORMATION FOR SEQ ID NO:27:
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention concerne la structure et la fonction d'un gène régulateur SCARECROW (SCR). Ce gène SCR est exprimé de façon spécifique dans les tissus parents radiculaires des embryons, ainsi que dans les racines et les tiges de semis et de plantes. L'expression de SCR commande la division cellulaire de certains types de cellules dans les racines et exerce une influence sur l'organisation de tissus de racines, ainsi que sur le géotropisme de structures aériennes. L'invention concerne également le gène SCARECROW (SCR), les produits du gène SCR (y compris, de façon non exhaustive, des produits de transcription, tels que ARNm, des molécules antisens et des molécules de ribozyme, et des produits de traduction, tels que la protéine de SCR, des polypeptides, des peptides et des protéines de fusion relatifs à ce gène), des anticorps dirigés contre SCR, des promoteurs de SCR et des zones de régulation, ainsi que l'utilisation de ce gène afin d'améliorer des plantes présentant un intérêt sur le plan agronomique.
EP97928623A 1996-04-26 1997-04-25 Gene scarecrow, son promoteur et ses utilisations Withdrawn EP0907660A4 (fr)

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US63861796A 1996-04-26 1996-04-26
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US08/842,445 US6441270B1 (en) 1996-04-26 1997-04-24 Scarecrow gene
PCT/US1997/007022 WO1997041152A1 (fr) 1996-04-26 1997-04-25 Gene scarecrow, son promoteur et ses utilisations

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AU2002228683A1 (en) 2000-11-29 2002-06-11 Albertus Wilhelm Martinus Bonke Wooden leg gene, promoter and uses thereof
WO2005014787A2 (fr) * 2003-07-17 2005-02-17 University Of Florida Elements genetiques conferant une expression de transgene specifique de petale de fleur
US9238818B2 (en) * 2004-04-20 2016-01-19 Syngenta Participations Ag Methods and genetic constructs for modification of lignin composition of corn cobs
EP2366789A1 (fr) * 2004-04-20 2011-09-21 Syngenta Participations AG Séquences de régulation pour exprimer des produits géniques dans des tissus de reproduction de plantes
DE602005017346D1 (de) 2004-12-08 2009-12-10 Sungene Gmbh Expressionskassetten für preferentiell vaskuläre Expression in Pflanzen
EP2163634A1 (fr) * 2004-12-08 2010-03-17 SunGene GmbH Cassettes d'expression pour expression préférentielle de tissu vasculaire dans des plantes
US20090307804A1 (en) * 2005-02-03 2009-12-10 Sungene Gmbh Transcription Regulating Nucleotide Sequence from Solanaceae Triose-Phosphate Translocator Genes and Their Use in Plant Expression Cassettes
EP2456874A1 (fr) 2009-07-24 2012-05-30 Pioneer Hi-Bred International Inc. Utilisation d'empilements de composants de domaines de dimérisation pour moduler l'architecture d'une plante
US8841434B2 (en) * 2009-09-30 2014-09-23 The United States Of America, As Represented By The Secretary Of Agriculture Isolated rice LP2 promoters and uses thereof
KR101383340B1 (ko) 2012-06-15 2014-04-10 건국대학교 산학협력단 애기장대 슛 시스템에서 성장 및 발달을 조절하는 방법
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RU2017124652A (ru) * 2015-01-06 2019-02-08 ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи Специфические для семян brassica napus промоторы, идентифицированные посредством анализа микромассивов
KR102034932B1 (ko) * 2017-10-25 2019-10-21 원광대학교산학협력단 생장양상이 신규한 유전적으로 변형된 토마토 식물체
WO2020097590A1 (fr) * 2018-11-09 2020-05-14 Duke University Compositions et procédés de modulation de la croissance des racines
CN110412263B (zh) * 2019-07-31 2023-01-31 中国农业科学院茶叶研究所 一种快速高效的茶树质体型谷氨酰胺合成酶基因定位方法
KR102101128B1 (ko) * 2019-10-14 2020-04-14 원광대학교산학협력단 생장양상이 신규한 유전적으로 변형된 토마토 식물체
CN111635955B (zh) * 2020-06-15 2023-05-09 中国科学院分子植物科学卓越创新中心 Shr-scr在豆科植物皮层细胞命运决定和改造非豆科植物皮层细胞分裂潜能中的应用
CN116284442B (zh) * 2023-02-08 2023-10-17 中国农业科学院生物技术研究所 一种控制叶片颜色的融合蛋白及其在植物转录因子与dna互作研究上的应用

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EP0907660A4 (fr) 2002-10-23
US20020032917A1 (en) 2002-03-14
US6455672B1 (en) 2002-09-24
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AU724857B2 (en) 2000-10-05

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